Supporting Information Synthesis and Initial Evaluation of Quinoline

Supporting Information Synthesis and Initial Evaluation of Quinoline-Based Inhibitors of the SH2-Containing Inositol 5'Phosphatase (SHIP) Christopher M. Russo,1 Arijit A. Adhikari,1 Daniel R. Wallach,1 Sandra Fernandes,2 Amanda N. Balch,2 William G. Kerr2 and John D. Chisholm*1 1

Department of Chemistry, Syracuse University, 1-014 Center for Science and Technology, Syracuse, NY, 13244-4100, USA

2

Department of Microbiology & Immunology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY, USA [email protected]

 

Table of Contents

S1

General Methods

S2

Experimental Procedures

S3-S14

1

S15-S39

H and 13C NMR Spectra

References

S40

S1  

General Information. All anhydrous reactions were run under a positive pressure of argon or nitrogen. All syringes, needles, and reaction flasks required for anhydrous reactions were dried in an oven and cooled under an argon atmosphere or in a desiccator. Dichloromethane, tetrahydrofuran, and diethyl ether were dried by passage through an alumina column following the method of Grubbs.1 Dimethylformamide was dried with 4Å molecular sieves and purified by vacuum distillation. All other reagents and solvents were purchased from commercial sources and used without further purification. Analysis and Purification. Analytical thin layer chromatography (TLC) was performed on precoated, glass-backed plates (silica gel 60 F 254 ; 0.25 mm thickness). The TLC plates were visualized by UV illumination and by staining. Solvents for chromatography are listed as volume:volume ratios. Flash column chromatography was carried out on silica gel (40-63 µm). Melting points were recorded using an electrothermal melting point apparatus and are uncorrected. Elemental analyses were performed on an elemental analyzer with a thermal conductivity detector and 2 meter GC column maintained at 50 °C. Identity. Proton (1H NMR) and carbon (13C NMR) nuclear magnetic resonance spectra were recorded at 300 or 400 MHz and 75 or 100 MHz respectively. The chemical shifts are given in parts per million (ppm) on the delta (δ) scale. Coupling constants are reported in hertz (Hz). The spectra were recorded in solutions of deuterated chloroform (CDCl3) with residual chloroform (δ 7.26 ppm for 1H NMR, δ 77.23 for 13C NMR) as the internal reference or in solutions of deuterated dimethyl sulfoxide (DMSO-d6) with residual dimethyl sulfoxide (δ 2.50 ppm for 1H NMR, δ 39.51 for 13C NMR) as the internal reference. Data are reported as follows: (s = singlet; d = doublet; t = triplet; q = quartet; p = pentet; sep = septet; dd = doublet of doublets; dt = doublet of triplets; td = triplet of doublets; tt = triplet of triplets; qd = quartet of doublets; ddd = doublet of doublet of doublets; br s = broad singlet). Where applicable, the number of protons attached to the corresponding carbon atom was determined by DEPT-135 NMR. Infrared (IR) spectra were obtained from thin films deposited on KBr plates by evaporation of a solution of the compound in CH2Cl2 or as a solid in a KBr pellet.

S2  

+ N

HO

H

H Cl -

N

4•HCl

2-(Hydroxy(2-phenylbenzo[h]quinolin-4-yl)methyl)piperidin-1-ium chloride 4•HCl.2 Amine 4 (1.37 g, 3.72 mmol) was suspended in 100 mL diethyl ether and anhydrous hydrogen chloride (2M in diethyl ether, 4.0 mL, 8.0 mmol) was added. The resulting precipitate was collected by vacuum filtration, suspended in 100 mL ethanol, and filtered again to provide 900 mg (60%) amine hydrochloride salt 4•HCl as a white solid. 4•HCl. mp = 258 °C (dec.); IR (KBr) 3280, 2936, 2854, 2710, 1589, 1377, 1106, 832, 698 cm-1; 1 H NMR (300 MHz, DMSO-d6) δ 9.65 (d, J = 12.8 Hz, 1H), 9.43-9.37 (m, 1H), 8.39 (d, J = 10.4 Hz, 3H), 8.32 (s, 1H), 8.24 (d, J = 9.6 Hz, 1H), 8.10-8.06 (m, 1H), 8.03 (d, J = 12.8 Hz, 1H), 7.84-7.75 (m, 2H), 7.65-7.58 (m, 2H), 7.57-7.50 (m, 1H), 6.60 (d, J = 4.2 Hz, 1H), 5.90 (s, 1H), 3.52-3.39 (m, 1H), 3.09-2.94 (m, 1H), 1.79-1.52 (m, 4H), 1.37-1.19 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 153.8, 147.3, 145.2, 138.7, 132.9, 131.0, 129.5, 128.9, 128.4, 127.8, 127.6, 127.1, 126.9, 124.3, 121.7, 120.9, 116.3, 67.5, 58.7, 44.0, 21.6, 21.1, 20.8. Anal. Calcd for C25H25ClN2O: C, 74.15; H, 6.22; N, 6.92. Found: C, 74.19; H, 5.95; N, 6.82. HO

4

N H N

Ph

(2-Phenylbenzo[h]quinolin-4-yl)(piperidin-2-yl)methanol 4. Epoxide 20 (3.00 g, 6.00 mmol) was dissolved in a mixture of 30 mL ethanol and 30 mL THF. Hydrazine hydrate (600 µL, 12.4 mmol) was added and the reaction was refluxed for 2 hours. The reaction was concentrated under vacuum to provide a crude mixture of product and byproducts. Silica gel chromatography (90% dichloromethane / 9% methanol / 1% NH4OH) provided the free amine as a brown solid with some orange colored impurities. The mixture was suspended in 30 mL methanol and filtered to provide amine 4 (1.37 g ,62%) as a tan solid. 4. mp = 175 °C (dec.); TLC Rf = 0.20 (90% dichloromethane / 9% methanol / 1% NH4OH); IR (KBr) 3286, 3060, 2932, 2851, 2744, 1590, 1443, 1382, 1107, 1048, 885, 700 cm-1; 1H NMR (300 MHz, DMSO-d6) δ 9.42- 9.38 (m, 1H), 8.41-8.38 (m, 2H), 8.30 (s, 1H), 8.16 (d, J = 9.3 Hz, 1H), 8.06-8.03 (m, 1H), 7.95 (d, J = 9.2 Hz, 1H), 7.82-7.73 (m, 2H), 7.65-7.59 (m, 2H), 7.567.50 (m, 1H), 5.73 (br s, 1H), 5.34 (br s, 1H), 2.96-2.86 (m, 2H), 2.54-2.40 (m, 1H), 1.74-1.66 (m, 1H), 1.59-1.51 (m, 1H), 1.48-1.40 (m, 1H), 1.34-1.14 (m, 3H); 13C NMR (100 MHz, DMSOd6) δ 153.8, 150.3, 145.3, 139.0, 133.0, 131.3, 129.4, 129.0, 128.3, 127.8, 127.0, 126.9, 124.5, 122.8, 121.6, 116.9, 72.2, 61.4, 46.5, 26.7, 26.0, 24.1 (One signal in the aromatic region was not resolved). Anal. Calcd for C25H24N2O: C, 81.49; H, 6.57; N, 7.60. Found: C, 81.16; H, 6.24; N, 7.83.

S3  

+ N

HO

H H Cl -

Cl N Cl

5•HCl

2-((2-(Adamantan-1-yl)-6,8-dichloroquinolin-4-yl)(hydroxy)methyl)piperidin-1-ium chloride 5•HCl.3 Amine 5 (678 mg, 1.52 mmol) was suspended in diethyl ether and anhydrous hydrogen chloride (2M in diethyl ether, 1.6 mL, 3.2 mmol) was added. The resulting precipitate was collected by vacuum filtration, re-suspended in 25 mL diethyl ether with 25 mL of ethanol added and filtered again to provide 440 mg (60%) of 5•HCl as a white solid. 5•HCl. mp = 202 °C (dec.); IR (film) 3271, 2904, 2847, 1595, 1451, 1130 cm-1; 1H NMR (300 MHz, DMSO-d6) δ 10.15 (d, J = 9.5 Hz, 1H), 8.52 (d, J = 2.1 Hz, 1H), 8.46-8.35 (m, 1H), 8.05 (d, J = 2.1 Hz, 1H), 7.85 (s, 1H), 6.57 (d, J = 4.5 Hz, 1H), 5.85 (s, 1H), 3.31-3.20 (m, 2H), 2.94 (q, J = 11.5 Hz, 1H), 2.12 (br s, 3H), 2.06 (br s, 6H), 1.78 (br s, 6H), 1.72-1.53 (m, 4H), 1.331.12 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.8, 146.4, 141.6, 134.6, 130.4, 129.4, 125.2, 121.9, 117.5, 67.6, 58.4, 44.1, 41.1, 39.8, 36.2, 28.0, 21.7, 21..0, 20.7. Anal. Calcd for C25H31Cl3N2O: C, 62.31; H, 6.48; N, 5.81. Found: C, 62.03 H, 6.36; N, 6.22. HO

N H

Cl

Cl

N 5

(2-(Adamantan-1-yl)-6,8-dichloroquinolin-4-yl)(piperidin-2-yl)methanol 5. Epoxide 30 (2.50 g, 4.34 mmol) was dissolved in 15 mL ethanol. Hydrazine hydrate (632 µL, 13.0 mmol) was added and the mixture was refluxed for 3 hours. After cooling to room temperature, the mixture was concentrated under vacuum and purified by silica gel chromatography (90% dichloromethane / 9% methanol / 1% NH4OH) to provide 983 mg of amine 5 (51%) as a beige solid. 5. mp = 180 °C (dec.); TLC Rf = 0.29 (90% dichloromethane / 9% methanol / 1% NH4OH); IR (KBr) 3309, 3068, 2906, 2849, 2675, 1597, 1482, 1309, 1116, 869 cm-1; 1H NMR (300 MHz, DMSO-d6) δ 8.25 (d, J = 2.1 Hz, 1H), 7.99 (d, J = 2.1, 1H), 7.78 (s, 1H), 5.66 (s, 1H), 5.08 (s, 1H), 2.88 (d, J = 14.4 Hz, 1H), 2.71-2.63 (m, 1H), 2.44-2.30 (m, 1H), 2.11 (br s, 3H), 2.05 (br s, 6H), 1.78 (br s, 6H), 1.73-1.64 (m, 1H), 1.48-1.38 (m, 2H), 1.28-1.11 (m, 3H); 13C NMR (100 MHz, DMSO-d6) δ 168.8, 149.1, 141.6, 134.5, 129.3, 128.9, 126.1, 122.4, 117.7, 72.4, 61.1, 46.3, 41.2, 41.1, 36.2, 28.0, 26.2, 25.8, 23.9. Anal. Calcd for C25H30Cl2N2O: C, 67.41; H, 6.79; N, 6.29. Found: C, 67.55; H, 6.96; N, 6.26. O

N O

4

HO

2-(5-Hydroxypentyl)isoindoline-1,3-dione. 5-Amino-1-pentanol (18.7 g, 181 mmol) and phthalic anhydride (26.8 g, 181 mmol) were heated to reflux in 181 mL toluene with a Dean-Stark condenser attached for 24 hours. The mixture was S4  

cooled to room temperature, transferred to a separatory funnel using ethyl acetate, washed once with brine, dried with sodium sulfate, and concentrated under vacuum to provide 39.2 g (93%) 2(5-hydroxypentyl)isoindoline-1,3-dione as a white solid. 2-(5-Hydroxypentyl)isoindoline-1,3-dione. mp = 43-48 °C; TLC Rf = 0.29 (60% ethyl acetate / 40% hexanes); 1H NMR (300 MHz, CDCl3) δ 7.87-7.80 (m, 2H), 7.74-7.67 (m, 2H), 3.70 (t, J = 7.1 Hz, 2H), 3.64 (t, J = 6.3 Hz, 2H), 1.77-1.56 (m, 4H), 1.48-1.36 (m, 2H). O

N O

O

13

5-(1,3-Dioxoisoindolin-2-yl)pentanal 13.4 2-(5-hydroxypentyl)isoindoline-1,3-dione (932 mg, 4.00 mmol) was dissolved in 13 mL anhydrous dichloromethane and cooled to 0°C while under argon. Trichloroisocyanuric acid (974 mg, 4.20 mmol) and TEMPO (6 mg, 0.04 mmol) were then added. The mixture was stirred at room temperature for 20 minutes and then filtered through celite. The resulting solution was washed with saturated sodium bicarbonate, 1M HCl, and brine, dried with sodium sulfate, and concentrated under vacuum to provide 850 mg (92%) aldehyde 13 as a yellow oil. 13. TLC Rf = 0.70 (100% diethyl ether); 1H NMR (300 MHz, CDCl3) δ 9.76 (t, J = 1.6 Hz, 1H), 7.88-7.81 (m, 2H), 7.75-7.68 (m, 2H), 3.71 (t, J = 6.8 Hz, 2H), 2.51 (td, J = 7.4, 1.5 Hz, 2H), 1.80-1.61 (m, 4H). O

OH

N

15

Ph

2-Phenylbenzo[h]quinoline-4-carboxylic acid 15.2 1-Naphthylamine 14 (25.0 g, 175 mmol) was dissolved in 100 mL of ethanol. Benzaldehyde (17.8 mL, 18.6 g, 175 mmol) and pyruvic acid (12.2 mL, 15.4 g, 175 mmol) were added sequentially to the 1-naphthylamine solution at room temperature. The reaction was then refluxed open to air for 3 h. After cooling to room temperature, the mixture was vacuum filtered. The resulting solid was thoroughly washed with ethanol and dried under vacuum to yield 13.7 g (26%) of carboxylic acid 15 as a yellow solid. 15. mp = 294-297 °C; TLC Rf = 0.11 (20% ethyl acetate / 80% hexanes); IR (KBr) 3061, 2623, 1704, 1256, 868, 742, 687 cm-1; 1H NMR (300 MHz, DMSO-d6) δ 9.42-9.39 (m, 1H), 8.58 (s, 1H), 8.54 (d, J = 9.3 Hz, 1H), 8.48-8.44 (m, 2H), 8.11-8.04 (m, 2H), 7.87-7.79 (m, 2H), 7.667.53 (m, 3H); 13C NMR (75 MHz, DMSO-d6) δ 167.9, 154.4, 146.3, 138.1, 138.0, 133.1, 130.8, 129.9, 129.1, 128.9, 128.7, 128.0, 127.5, 127.2, 124.5, 122.3, 121.9, 118.9. Anal. Calcd for C20H13NO2: C, 80.25; H, 4.38; N, 4.68;. Found: C, 80.04; H, 4.39; N, 4.96.

S5  

HO

N

16

Ph

(2-Phenylbenzo[h]quinolin-4-yl)methanol 16. Carboxylic acid 15 (5.00 g, 16.7 mmol) was suspended in 17 mL anhydrous THF under argon. After cooling the mixture to 0 °C, borane-tetrahydrofuran complex (1M in THF, 34.0 mL, 34.0 mmol) was slowly added to the well-stirred mixture. After hydrogen gas evolution ceased, the reaction was allowed to warm to room temperature and stirred for 6 h. The mixture was then cooled to 0 °C and aqueous NaOH (3 M, 17 mL) was slowly added. The mixture was stirred at room temperature for 12 h, after which the THF was removed under vacuum. The resulting mixture was then extracted with ethyl acetate. The combined organic extracts were dried with MgSO4, filtered, and evaporated to an oil, which was diluted with 50 mL of methanol. The methanol was then removed under vacuum to yield 3.81 g (80%) of alcohol 16 as a white solid. 16. mp = 140-144 °C; TLC Rf = 0.32 (20% ethyl acetate / 80% hexanes); IR (KBr) 3343, 3061, 2899, 1591, 1377, 1020, 756, 693 cm-1; 1H NMR (300 MHz, DMSO-d6) δ 9.41-9.38 (m, 1H), 8.44-8.40 (m, 2H), 8.32 (s, 1H), 8.03-8.07 (m, 1H), 8.00-7.94 (m, 2H), 7.83-7.74 (m, 2H), 7.657.59 (m, 2H), 7.56-7.50 (m, 1H), 5.72 (t, J = 5.5 Hz, 1H), 5.16 (d, J = 5.5 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) δ 154.4, 149.0, 145.0, 139.1, 133.2, 131.3, 129.5, 129.0, 128.3, 128.0, 127.13, 127.09, 127.05, 124.4, 122.5, 121.1, 116.0, 60.3. Anal. Calcd. for C20H15NO: C, 84.19; H, 5.30; N, 4.91. Found: C, 84.06; H, 4.97; N, 5.09. Cl

N

17

Ph

4-(Chloromethyl)-2-phenylbenzo[h]quinoline 17. Alcohol 16 (13.5 g, 47.4 mmol) was dissolved in 95 mL anhydrous dichloromethane. Neat thionyl chloride (4.82 mL, 7.90 g, 66.4 mmol) was slowly added to the solution at room temperature. The reaction was stirred at room temperature for 18 h and then carefully quenched with saturated aqueous NaHCO3. The reaction was diluted with an equal volume of water and vigorously stirred until all solids dissolved. The organic layer was separated, washed with brine, dried with Na2SO4, filtered, and evaporated under vacuum to yield 10.3 g (72%) of chloride 17 as a tan solid. 17. mp = 145-153 °C; TLC Rf = 0.80 (20% ethyl acetate / 80% hexanes); IR (KBr) 3062, 2942, 1588, 1053, 752, 689 cm-1; 1H NMR (400 MHz, CDCl3) δ 9.53-9.50 (m, 1H), 8.36-8.34 (m, 2H), 8.06 (s, 1H), 7.99 (d, J = 8.7 Hz, 1H), 7.95-7.88 (m, 2H), 7.79-7.70 (m, 2H), 7.60-7.55 (m, 2H), 7.52-7.48 (m, 1H), 5.10 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 155.4, 146.8, 142.5, 139.4, 133.7, 132.2, 129.6, 129.0, 128.6, 128.3, 127.9, 127.6, 127.3, 125.3, 123.0, 120.4, 119.1, 43.1. Anal. Calcd. for C20H14ClN: C, 79.07; H, 4.65; N, 4.61. Found: C, 78.96; H, 4.50; N 4.83.

S6  

O MeO P MeO

N

18

Ph

Dimethyl ((2-phenylbenzo[h]quinolin-4-yl)methyl)phosphonate 18 Chloride 17 (1.97 g, 6.50 mmol) and trimethyl phosphite (13.7 g, 13.0 mL, 110 mmol) were combined in a flask with 6.5 mL toluene. The mixture was refluxed for 3 d. After cooling to room temperature, the mixture was concentrated under vacuum to a brown oil which was triturated with diethyl ether. The resulting precipitate was vacuum filtered and washed with cold diethyl ether to yield 2.12 g (87%) of phosphonate 18 as a white solid. 18. mp = 136-137 °C; TLC Rf = 0.43 (20% acetone / 80% dichloromethane); IR (KBr) 3061, 2953, 2851, 1587, 1252, 1053, 804 cm-1; 1H NMR (300 MHz, CDCl3) δ 9.54-9.51 (m, 1H), 8.378.34 (m, 2H), 8.02-7.84 (m, 4H), 7.79-7.68 (m, 2H), 7.60-7.54 (m, 2H), 7.52-7.46 (m, 1H), 3.75 (d, J = 22.5 Hz, 2H), 3.66 (d, J = 10.8 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 154.9 (d, J = 3.6 Hz), 146.8 (d, J = 2.5 Hz), 139.5, 138.4 (d, J = 9.3 Hz), 133.7, 132.2, 129.5, 129.0, 128.5, 127.79, 127.78, 127.6, 127.2, 125.3, 124.2 (d, J = 5.1 Hz), 121.2 (d, J = 1.5 Hz), 121.0 (d, J = 6.5 Hz), 53.3 (d, J = 6.7 Hz), 30.3 (d, J = 138.2 Hz). Anal. Calcd for C22H20NO3P: C, 70.02; H, 5.34; N, 3.71. Found C, 69.69; H, 5.42; N, 3.88. PhthN

N Ph 20 >20:1 E:Z

(E)-2-(6-(2-Phenylbenzo[h]quinolin-4-yl)hex-5-en-1-yl)isoindoline-1,3-dione 20. Phosphonate 18 (377 mg, 1.00 mmol) and lithium chloride (63 mg, 1.50 mmol) were combined in a flask under argon and 5 mL of anhydrous THF was added followed by DBU (228 mg, 224 µL, 1.50 mmol). Aldehyde 13 (347 mg, 1.50 mmol) was dissolved in 5 mL anhydrous THF under argon. The aldehyde solution was then transferred to the stirred phosphonate solution dropwise at room temperature. After stirring for 24 hours, the reaction was diluted with water and extracted with ethyl acetate. The combined organic extracts were dried with MgSO4, filtered, and concentrated under vacuum. Purification by silica gel chromatography (20% ethyl acetate / 80% hexanes) provided 330 mg (68%) of olefin 20 as a yellow solid. 20. mp = 135-144 °C; TLC Rf = 0.67 (30% ethyl acetate / 70% hexanes); IR (KBr) 3059, 2934, 2872, 1705, 1581, 1398, 1368, 1036, 721 cm-1; 1H NMR (300 MHz, CDCl3) δ 9.52-9.50 (m, 1H), 8.37-8.35 (m, 2H), 8.02 (s, 1H), 8.00 (d, J = 9.2 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.86-7.82 (m, 2H), 7.80 (d, J = 9.1 Hz, 1H), 7.76-7.67 (m, 4H), 7.59-7.54 (m, 2H), 7.50-7.46 (m, 1H), 7.19 (d, J = 15.7 Hz, 1H), 6.53 (dt, J = 15.7, 6.9 Hz, 1H), 3.79 (t, J = 7.3 Hz, 2H), 2.47 (q, J = 7.1 Hz, 2H), 1.85 (p, J = 7.5 Hz, 2H), 1.67 (p, J = 7.6 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 168.6, 155.1, 146.6, 144.0, 140.1, 137.1, 134.0, 133.7, 132.21, 132.19, 129.2, 128.9, 128.1, 127.7, 127.6, 127.1, 126.9, 126.1, 125.3, 123.3, 122.9, 121.2, 115.6, 37.9, 33.1, 28.3, 26.4. Anal. Calcd for C33H26N2O2: C, 82.13; H, 5.43; N, 5.81;. Found: C, 82.44; H, 5.13; N, 5.84.

S7  

PhthN

O

Ph

N

21

2-(4-(3-(2-Phenylbenzo[h]quinolin-4-yl)oxiran-2-yl)butyl)isoindoline-1,3-dione 21. Olefin 21 (4.76 g, 9.86 mmol) was dissolved in 62 mL of CHCl3. To this solution was added 3chloroperbenzoic acid (approx. 70%, 5.11 g, approx. 29.6 mmol). The mixture was refluxed for 18 h. Excess peracid was consumed by vigorously stirring the reaction with 10% aq. Na2SO3. The mixture was poured into sat. aq. NaHCO3 and extracted with dichloromethane. The combined organic extracts were washed once with brine, dried with Na2SO4, and concentrated under vacuum. The crude solid was triturated with methanol and filtered to provide 3.00 g of epoxide 21 (61%) as a tan solid. 21. mp = 55-69 °C; TLC Rf = 0.61 (30% ethyl acetate / 70% hexanes); IR (KBr) 3059, 2933, 2858, 1710, 1590, 1466, 720 cm-1; 1H NMR (400 MHz, CDCl3) δ 9.51 (dd, J = 7.9, 0.8 Hz, 1H), 8.37-8.34 (m, 2H), 7.95 (s, 1H), 7.93-7.89 (m, 2H), 7.88-7.83 (m, 2H), 7.80 (d, J = 9.0 Hz, 1H), 7.78-7.69 (m, 4H), 7.58-7.52 (m, 2H), 7.50-7.45 (m, 1H), 4.36 (d, J = 1.9 Hz, 1H), 3.78 (t, J = 7.2 Hz, 2H), 3.04-3.01 (m, 1H), 2.07-1.98 (m, 1H), 1.95-1.80 (m, 3H), 1.75-1.62 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 168.6, 155.7, 146.0, 144.0, 139.7, 134.2, 133.6, 132.3, 132.2, 129.5, 129.0, 128.4, 127.93, 127.90, 127.7, 127.3, 125.4, 123.4, 123.2, 120.0, 114.2, 62.9, 55.9, 37.8, 32.1, 28.6, 23.5. Anal. Calcd for C33H26N2O3: C, 79.50; H, 5.26; N, 5.62. Found: C, 79.46; H, 5.28; N, 5.65. O

Cl

O

Cl

5

N H

23

5,7-Dichloroindoline-2,3-dione 23. Isatin (14.7 g, 100 mmol) and trichloroisocyanuric acid (23.2 g, 100 mmol) were combined in a flask and cooled to -78°C with a dry ice-acetone bath. Concentrated sulfuric acid (75 mL) was added dropwise to the mixture via addition funnel. The mixture was allowed to slowly warm to room temperature. After stirring for 3 days, the mixture was poured over ice and stirred until all the ice had melted. The precipitate was collected by vacuum filtration and washed twice with water. The orange-red solid was then washed with acetone until only a white solid (isocyanuric acid) remained. The filtrate was concentrated under vacuum to yield 19.8 g (92%) of dichloroisatin 23 as an orange-red solid. 23. mp = 211-217 °C; TLC Rf = 0.65 (50% ethyl acetate / 50% hexanes); 1H NMR (300 MHz, DMSO-d6) δ 11.59 (br s, 1H), 7.85 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 2.0 Hz, 1H). O

25

3

1-(Adamantan-1-yl)ethanone 25. 1-Adamantanecarboxylic acid (7.20 g, 40.0 mmol) was dissolved in 40 mL of diethyl ether under argon. The mixture was maintained at approximately -5 °C with a NaCl-ice bath while methyl lithium (1.6M in diethyl ether, 52.5 mL, 84.0 mmol) was added dropwise with vigorous stirring. After complete addition, the cooling bath was removed and the slurry was allowed to stir for one S8  

hour at room temperature. The reaction was quenched and diluted by the addition of water and extracted with diethyl ether. The combined organic extracts were dried with MgSO4, filtered, concentrated under vacuum, and purified by silica gel chromatography (10% ethyl acetate / 90% hexanes) to yield 5.70 g (80%) of ketone 25 as a white solid. 25. mp = 52-54 °C; TLC Rf = 0.43 (10% ethyl acetate / 90% hexanes); 1H NMR (300 MHz, CDCl3) δ 2.09 (s, 3H), 2.04 (br s, 3H), 1.82-1.64 (m, 12H). O

OH

Cl N Cl

26

2-(Adamantan-1-yl)-6,8-dichloroquinoline-4-carboxylic acid 26.3 Dichloroisatin 23 (33.8 g, 157 mmol, 2.0 equiv), ketone 25 (14.0 g, 78.7 mmol, 1.0 equiv), and potassium hydroxide (28.6 g, 501 mmol, 6.4 mmol) were combined with 78 mL ethanol and 26 mL H2O and heated to reflux for 48 hours. The mixture was then allowed to cool to room temperature and concentrated under vacuum to leave a brown paste, which was taken up in H2O and diethyl ether. The organic layer was discarded and the aqueous layer was washed once more with diethyl ether. The aqueous layer was then acidified (pH 4 to 5) by the dropwise addition of concentrated HCl. The resulting precipitate was isolated by vacuum filtration and recrystallized from ethanol to provide 12.7 g (43%) of carboxylic acid 26 as a tan solid. 26. mp = 152-157 °C; TLC Rf = 0.15 (30% ethyl acetate / 70% hexanes); IR (KBr) 3462, 2902, 2848, 2651, 1703, 1591, 1268, 1193 cm-1; 1H NMR (300 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.168.12 (m, 2H), 2.68 (br s, 6H), 2.11 (br s, 3H), 1.79 (br s, 6H); 13C NMR (100 MHz, DMSO-d6) δ 169.4, 166.9, 142.2, 136.1, 134.5, 131.1, 129.7, 124.6, 123.6, 120.8, 40.9, 36.0, 28.0 (One signal in the aliphatic region was not resolved). Anal. Calcd for C20H19Cl2NO2: C, 63.84; H, 5.09; N, 3.72. Found: C, 64.18; H, 5.03; N, 3.87. OH Cl

Cl

N 27

2-(Adamantan-1-yl)-6,8-dichloroquinolin-4-yl)methanol 27. Carboxylic acid 26 (3.48 g, 9.26 mmol) was dissolved in 10 mL of anhydrous THF under argon. After cooling the mixture to 0°C, borane-tetrahydrofuran complex (1 M in THF, 18.5 mL, 18.5 mmol) was slowly added to the mixture. After hydrogen gas evolution ceased, the cooling bath was removed and the mixture was allowed to stir at room temperature overnight. The mixture was again cooled to 0°C and quenched with 20 mL 3M NaOH. The cooling bath was removed and, after stirring at room temperature for 6 hours, the mixture was extracted with diethyl ether. The combined organic extracts were dried with MgSO4, filtered, concentrated under vacuum, and purified by silica gel chromatography (30% ethyl acetate / 70% hexanes) to yield 2.00 g (60%) of alcohol 27 as a beige solid. 27. mp = 190-198 °C; TLC Rf = 0.56 (30% ethyl acetate / 70% hexanes); IR (KBr) 3278, 2899, 2845, 1596, 1448, 1081, 1060 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.83 (d, J = 2.3 Hz, 1H), 7.77 (d, J = 2.2 Hz, 1H), 7.68 (s, 1H), 5.13 (dd, J = 5.7, 0.9 Hz, 2H), 2.16 (br s, 3H), 2.13 (br s, 6H), 1.91 (t, J = 5.8 Hz, 1H), 1.83 (br s, 6H); 13C NMR (75 MHz, CDCl3) δ 170.1, 145.1, 142.5, S9  

135.9, 130.9, 129.7, 126.0, 121.1, 117.0, 62.3, 41.9, 40.6, 37.0, 28.9. Anal. Calcd. for C20H21Cl2NO: C, 66.30; H, 5.84; N, 3.87. Found: C, 66.44; H, 5.59; N, 3.69. Cl Cl N Cl

28

2-(Adamantan-1-yl)-6,8-dichloro-4-(chloromethyl)quinoline 28. Alcohol 27 (8.36 g, 23.1 mmol) was dissolved in dichloromethane (250 mL). Thionyl chloride (2.52 mL 34.66 mmol) was then added. The mixture was stirred at 0 °C for 1 h and then quenched with sat. aq. NaHCO3. The organic layer was separated and the aqueous layer was extracted with dichloromethane. The combined organic extracts were dried over sodium sulfate, filtered, concentrated under vacuum, and purified by silica gel chromatography (1% ether / 99% hexanes) to provide 4.40 g (50%) of chloride 28 as a white solid. 28. mp = 155-158 °C; TLC Rf = 0.34 (100% hexanes); IR (KBr) 2900, 2847, 1672, 1597, 1450, 721 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.94 (d, J = 2.2 Hz, 1H), 7.80 (d, J = 2.4 Hz, 1H), 7.58 (s, 1H), 4.91 (s, 2H), 2.17 (br s, 3H), 2.12 (br s, 6H), 1.83 (br s, 6H); 13C NMR (75 MHz, CDCl3) δ 170.0, 142.9, 141.5, 136.2, 131.4, 130.1, 126.1, 121.3, 119.9, 42.8, 41.8, 40.6, 36.9 28.8. Anal. Calcd. for C20H20Cl3N: C, 63.09; H, 5.29; N, 3.68. Found: C, 63.28; H, 5.63; N, 3.57. O P OMe OMe Cl N Cl

29

Dimethyl ((2-(adamantan-1-yl)-6,8-dichloroquinolin-4-yl)methyl)phosphonate 29. Chloride 28 (1.20 g, 3.15 mmol) and trimethyl phosphite (6.27 mL, 53.2 mmol) were combined in a flask with 3.5 mL toluene under argon. The mixture was refluxed for 3 days, concentrated under vacuum, and purified by silica gel chromatography (40% ethyl acetate / 60% hexanes) to provide 1.06 g (74%) phosphonate 29 as a white solid. 29. mp = 152-153 °C; TLC Rf = 0.33 (60% ethyl acetate / 40% hexanes); IR (KBr) 2952, 2902, 2847, 1772, 1711, 1594, 1398, 1239, 1075, 1031, 856, 830 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.91 (d, J = 2.1 Hz, 1H), 7.78 (d, J = 2.1 Hz, 1H), 7.54 (d, J = 3.6 Hz, 1H), 3.66 (d, J = 11.1 Hz, 6H), 3.55 (d, J = 22.5 Hz, 2H), 2.16 (br s, 3H), 2.11 (br s, 6H), 1.82 (br s, 6H); 13C NMR (75 MHz, CDCl3) δ 169.4 (d, J = 3.9 Hz), 142.9 (d, J = 2.3 Hz), 137.5 (d, J = 9.2 Hz), 135.9, 131.0, 129.8, 127.4 (d, J = 4.7 Hz), 122.0 (d, J = 1.3 Hz), 121.7 (d, J = 7.0 Hz), 53.3 (d, J = 6.9 Hz), 41.8, 40.4, 36.9, 30.1 (d, J = 139.1 Hz), 28.8. Anal. Calcd. for C22H26Cl2NO3P: C, 58.16; H, 5.77; N, 3.08. Found: C, 57.97; H, 5.57; N, 2.94.

S10  

O N Cl

O

N Cl 30 >20:1 E:Z

2-((E)-6-(2-(Adamantan-1-yl)-6,8-dichloroquinolin-4-yl)hex-5-en-1-yl)isoindoline-1,3-dione 30. Phosphonate 29 (454 mg, 1.00 mmol) and lithium chloride (63 mg, 1.50 mmol) were combined in a flask under argon. Anhydrous THF (5 mL) was then added followed by DBU (152 mg, 149 µL, 1.00 mmol). Aldehyde 13 (347 mg, 1.50 mmol) was dissolved in 5 mL anhydrous THF under argon in a separate flask. The aldehyde solution was then transferred to the stirred phosphonate solution dropwise at room temperature. After stirring for 24 hours, the reaction was diluted with H2O and extracted with ethyl acetate. The combined organic extracts were dried with MgSO4, filtered, and concentrated under vacuum. Purification by silica gel chromatography (10 % ethyl acetate / 90% hexanes) provided 402 mg of olefin 30 (68 %) as a white foam. 30. mp = 63-69 °C; TLC Rf = 0.29 (5% ethyl acetate / 95% hexanes); IR (film) 2903, 2848, 1771, 1712, 1396, 719 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.92 (d, J = 2.1 Hz, 1H), 7.86-7.83 (m, 2H), 7.74-7.69 (m, 3H), 7.53 (s, 1H), 6.95 (d, J = 15.6 Hz, 1H), 6.40 (dt, J = 6.9, 15.6 Hz, 1H), 3.77 (t, J = 6.9 Hz, 2H), 2.42 (q, J = 6.9 Hz, 2H), 2.19-2.10 (m, 9H), 1.86-1.76 (m, 8H), 1.681.56 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 169.7, 168.6, 143.1, 142.8, 137.8, 135.6, 134.1, 132.2, 130.3, 129.5, 126.4, 125.4, 123.4, 121.8, 115.8, 41.9, 40.4, 37.8, 37.0, 33.1, 28.9, 28.3, 26.4. Anal. Calcd for C33H32Cl2N2O2: C, 70.84; H, 5.76; N, 5.01. Found: C, 70.57; H, 5.90; N, 5.03. O N

O

O Cl

Cl

N 31

2-(4-(3-(2-(Adamantan-1-yl)-6,8-dichloroquinolin-4-yl)oxiran-2-yl)butyl)isoindoline-1,3dione 31. Olefin 30 (3.27 g, 5.84 mmol) and 3-chloroperbenzoic acid (approx. 70%, 3.03 g, approx. 17.5 mmol) were dissolved in 34 mL of chloroform. The mixture was refluxed for 15 hours. After cooling to room temperature, the reaction was quenched with 10% aqueous Na2SO3, diluted with dichloromethane, and then washed with saturated sodium bicarbonate and brine. The organic layer was dried with sodium sulfate, filtered, concentrated under vacuum and purified by silica gel chromatography (10% ethyl acetate / 90% hexanes) to provide 2.52 g (75%) epoxide 31 as a white foam. 31. TLC Rf = 0.26 (30% ethyl acetate / 70% hexanes); IR (KBr) 2904, 2849, 1772, 1713, 1596, 1397, 720 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.87-7.83 (m, 3H), 7.77 (d, J = 2.1 Hz, 1H), 7.747.70 (m, 2H), 7.48 (s, 1H), 4.14 (d, J = 1.8 Hz, 1H), 3.76 (t, J = 7.1 Hz, 2H), 2.93-2.88 (m, 1H), 2.14 (br s, 3H), 2.10-1.96 (m, 8H), 1.88-1.76 (m, 8H), 1.70-1.58 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 170.3, 168.6, 142.9, 142.2, 136.1, 134.1, 132.2, 131.0, 129.7, 126.4, 123.4, 120.6, S11  

114.9, 62.9, 55.5, 41.8, 40.6, 37.7, 36.9, 31.9, 28.9, 28.5, 23.4. Anal. Calcd for C33H32Cl2N2O3: C, 68.87; H, 5.60; N, 4.87. Found: C, 68.98; H, 5.84; N, 4.92. +H

Cl -

3N

N Ph 34•HCl

(2-Phenylbenzo[h]quinolin-4-yl)methanaminium chloride 34•HCl. O Cl

N O

phthalimide, K 2CO3 N 17

Ph

DMF, 80 °C

N A 83% +H

H 2N

Cl -

NH 2NH 2•H 2O EtOH, reflux

Ph

3N

HCl N 34 90%

Ph

Et 2O

N 34•HCl 92%

Ph

Chloride 17 (304 mg, 1.00 mmol), phthalimide (177 mg, 1.20 mmol), and potassium carbonate (276 mg, 2.00 mmol) were combined with 10 mL DMF and heated to 80 °C for 24 hours. The mixture was cooled to room temperature and decanted to remove inorganic solids. The organic portion was diluted with 40 mL methanol to produce a precipitate, which was filtered, washed with methanol, and dried to provide 344 mg (83%) of quinolyl phthalimide A as a tan solid. A. mp = 215-218 °C; TLC Rf = 0.26 (20% ethyl acetate / 80% hexanes); IR (KBr) 2922, 2851, 1714, 1642, 1391, 1105 cm-1; 1H NMR (400 MHz, CDCl3) δ 9.52-9.50 (m, 1H), 8.31-8.25 (m, 3H), 8.07 (s, 1H), 7.93-7.85 (m, 4H), 7.78-7.68 (m, 4H), 7.56-7.51 (m, 2H), 7.48-7.44 (m, 1H), 5.42 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 168.2, 155.5, 146.9, 141.6, 139.8, 134.5, 133.7, 132.3, 132.1, 129.5, 129.0, 128.5, 128.2, 127.9, 127.8, 127.2, 125.4, 123.8, 123.3, 120.7, 119.6, 39.1. Anal. Calcd for C28H18N2O2: C, 81.14; H, 4.38; N, 6.76. Found: C, 80.79; H, 4.57; N, 6.76. The quinolyl phthalimide A (300 mg, 0.72 mmol) was suspended in 4 mL ethanol. Hydrazine hydrate (175 µL, 3.6 mmol) was added and the mixture was heated to reflux for 3 hours. The reaction was then cooled to room temperature and vacuum filtered. The resulting precipitate was washed with methanol and the combined filtrates were concentrated and purified by silica gel chromatography (5% methanol / 90% dichloromethane) to provide 184 mg of quinolyl amine 34 (90%) as a white solid. 34. mp = 121-123 °C; TLC Rf = 0.19 (5% methanol / 95% dichlormethane); IR (KBr) 3446, 3058, 1625, 1591, 1553, 1499, 1381 cm-1; 1H NMR (400 MHz, CDCl3) δ 9.53 (d, J = 8.2 Hz, 1H), 8.39-8.36 (m, 2H), 8.10 (s, 1H), 7.94-7.91 (m, 2H), 7.84 (d, J = 9.1 Hz, 1H), 7.78-7.68 (m, 2H), 7.59-7.55 (m, 2H), 7.52-7.46 (m, 1H), 4.48 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 155.6, 148.7, 146.4, 140.1, 133.7, 132.4, 129.4, 129.0, 128.3, 127.9, 127.7, 127.1, 125.4, 123.3, 120.4, 116.8, 43.4 (One signal in the aromatic region was not resolved). Anal. Calcd for C20H16N2: C, 84.48; H, 5.67; N, 9.85. Found: C, 84.44; H, 5.54; N, 9.61.

S12  

The quinolyl amine 34 (128 mg, 0.45 mmol) was dissolved in 3 mL methanol and HCl in diethyl ether (2 M, 270 µL, 0.54 mmol) was added. The mixture was stirred for 5 minutes and concentrated in vacuo. The resulting oil was triturated with diethyl ether until a yellow precipitate formed. This was collected by vacuum filtration, washed with diethyl ether, and dried to provide 133 mg (92%) amine hydrochloride 34•HCl as a yellow solid. 34•HCl. mp = 178 °C (dec.); IR (KBr) 3429, 3080, 2911, 1625, 1513, 1377 cm-1; 1H NMR (300 MHz, DMSO-d6) δ 9.42-9.38 (m, 1H), 8.80 (br s, 3H), 8.48-8.45 (m, 3H), 8.12-8.03 (m, 3H), 7.90-7.76 (m, 2H), 7.66-7.52 (m, 3H), 4.74 (q, J = 5.7 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δ 154.2, 145.3, 140.9, 138.6, 133.2, 131.0, 129.8, 129.0, 128.6, 128.0, 127.8, 127.4, 127.2, 124.4, 122.6, 120.9, 118.4, 38.7. Anal. Calcd for C20H17ClN2: C, 74.88; H, 5.34; N, 8.73. Found: C, 74.49; H, 5.40; N, 8.61. Malachite Green Phosphatase Release Assays Malachite Green Phosphatase Release Assays (Echelon-Inc.) were performed with recombinant human truncated SHIP1 (tSHIP1)6,7 or tSHIP2 (see below) as described previously.8 Briefly, serial dilutions of the compounds were dissolved in appropriate solvent (e.g. DMSO) and added to recombinant human tSHIP1 or tSHIP2 diluted in enzyme reaction buffer (50 mM Hepes pH 7.4, 150mM NaCl, 1mM MgCl2, 0.25mM EDTA) in triplicate reactions in 96-well plates (22.5 ul/reaction). Reactions were incubated for 5 min at room temperature. Phosphatidylinositol 3,4,5-trisphosphate diC8 (PI(3,4,5)P3diC8) (Echelon-Inc.) was added to each reaction at a final concentration of 100 µM and the reaction were carried out for 30 min at 37 ˚C in a final volume of 25 ul/well. Final solvent concentration was ≤ 1%. Following incubation, 100 µL of Malachite Green Solution (Echelon-Inc.) was added to each well and plates were incubated at room temperature in the dark for 20 min. Plates were then read at 620nm. Expression and purification of recombinant human truncated SHIP2 (tSHIP2). Human cDNA was obtained by random primed RT-PCR (Multiscribe Reverse Transcriptase, Invitrogen) on 100ng of total RNA extracted from human whole blood using QIAamp RNA blood Mini Kit (QIAGEN). Truncated SHIP2 (tSHIP2, nucleotides 199-3102, NM_001567.3) was amplified from cDNA with Phusion High-Fidelity DNA polymerase (New England Biolabs) in two fragments using forward and reverse primers 5’ATAGGATCCTATGGCCCCCTCCTGGTA-3 or 5’GCTCAATGCCTTTGACATCTCTCTGCG-3’, and 5’TGCAGATGATGTGAGTTTCAGGGTAGGA-3 or 5’GCGTAAGCTTGGCAGGGTTATTGAAGC-3’, respectively. The fragments were cloned into pSMART HC Amp (Lucigen) for assembly using the BamHI and HindIII restriction sites introduced by the primers and the natural NdeI site found in the INPPL1 sequence. The tSHIP2 coding sequence was then subcloned using BamHI and HindIII sites into the pET24-TEV vector6,7 using standard cloning procedures, and verified by sequencing. Restriction and DNA modifying enzymes were from New England Biolabs, primers from Integrated DNA technologies and sequencing was performed by Molecular Cloning Labs. E.coli BL21 DE3 (EMD Millipore) were used for expression of pET24TEV-tSHIP2 by 20h stationary-phase S13  

induction at 37˚C, 200rpm, in 1L LB Broth (Fisher) supplemented with 0.2% (w/v) D-Glucose (Sigma) and 50ug/ml kanamycin (Sigma) following 1:100 inoculation with starter culture. Protein was extracted form bacterial cell pellet using BugBuster HT (EMD Millipore), according to manufacturer’s recommendations. Protein was purified by FPLC using HisTrapHP 5ml column (GE) at a 5ml/min flow rate and 20mM-1M imidazole gradient in Buffer A (20mM Tris pH 8.0, 300mM NaCl, 0.1mM β-ME). Fractions containing active protein by Malachite Green Phosphatase Release Assay (Echelon, see below) were pooled and dialyzed for 16h at 4˚C in Buffer A + 0.1% Triton-X100. Dialyzed protein was concentrated 10-fold with Amicon Ultra-0.5 Ultracel 100 membrane (EMD Millipore). Protein was aliquoted and stored in Buffer A containing 50% glycerol at -20 ˚C or -80 ˚C for longer-term storage. tSHIP2 protein expressed from this vector contains the pET-24b leader sequences, amino acids 18-985 of human SHIP2 (INPPL1 NM_001567.3), TEV cleavage site and the 6-HIS-tag, resulting in a 1003 aa protein with a MW of 112kDa.

S14  

+ N

HO

H

H Cl -

N

NSC13480 (4•HCl) obtained from the NCI

S15  

+ N

HO

H

H Cl -

N

4•HCl

+ N

HO

H

H Cl -

N

4•HCl

S16  

HO

4

HO

4

N H N

N H N

Ph

S17  

Ph

+ N H H Cl -

HO Cl N Cl

NSC305787 (5•HCl) obtained from the NCI

S18  

+ N H H Cl -

HO Cl N Cl

5•HCl

+ N H H Cl -

HO Cl N Cl

5•HCl

S19  

HO

N H

Cl

Cl

N 5

HO

N H

Cl

Cl

N 5

S20  

O

N O

HO

S21  

O

N O

O

13

S22  

O

OH

N

15

Ph

O

OH

N

15

S23  

Ph

HO

N

16

Ph

HO

N

16

S24  

Ph

Cl

N

17

Cl

N

17

S25  

Ph

Ph

O MeO P MeO

N

18

Ph

O MeO P MeO

N

S26  

18

Ph

PhthN

N Ph 20 >20:1 E:Z

PhthN

N Ph 20 >20:1 E:Z

S27  

PhthN

O

N

Ph

21

PhthN

O

N

21

S28  

Ph

O

Cl

O

Cl

S29  

N H

23

O

25

S30  

O

OH

Cl N Cl

26

O

OH

Cl N Cl

26

S31  

OH Cl

Cl

N 27

OH Cl

Cl

N 27

S32  

Cl Cl N Cl

28

Cl Cl N Cl

28

S33  

O P OMe OMe Cl N Cl

29

O P OMe OMe Cl N Cl

29

 

S34  

O N O

Cl

N Cl 30 >20:1 E:Z

O N Cl

O

N Cl 30 >20:1 E:Z

S35  

O N

O

O Cl

Cl

N 31

O N

O

O Cl

Cl

N 31

S36  

S37  

H 2N

Ph

N 34

H 2N

N 34

S38  

Ph

+H

Cl -

3N

N Ph 34•HCl

+H

Cl -

3N

N Ph 34•HCl

S39  

References 1. Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. Safe and Convenient Procedure for Solvent Purification. Organometallics. 1996, 15, 1518. 2. Buchman, E. R.; Howton, D. R. Potential antimalarials. (2-Phenyl-7,8-benzo-4-quinolyl)2-piperidylcarbinols. J. Org. Chem. 1949, 14, 895-899. 3. Novotny, J.; Collins, C. H.; Starks, F. W. Synthesis and screening of potential antimalarial agent alpha -(2-piperidyl)-2-(1-adamantyl)-6,8-dichloro-4quinolinemethanol hydrochloride. J. Pharm. Sci. 1974, 63, 1264-1267. 4. Allegretti, P. A.; Ferreira, E. M. Vicinal Bisheterocyclizations of Alkynes via Nucleophilic Interception of a Catalytic Platinum Carbene. J. Am. Chem. Soc. 2013, 135, 17266-17269. 5. Ribeiro, N. M.; Da Silva, B. V.; de Almeida Violante, F.; Rezende, C. M.; Pinto, A. C. 5Chloro- and 5,7-dichloroisatin by chlorination of isatin with trichloroisocyanuric acid. Org. Prep. Proc. Int. 2005, 37, 265-267. 6. Brooks, R.; Iyer, S.; Akada, H.; Neelam, S.; Russo, C. M.; Chisholm, J. D.; Kerr, W. G., Coordinate expansion of murine hematopoietic and mesenchymal stem cell compartments bv SHIPi. Stem Cells 2015, 33, 848-858. 7. Blanco-Menendez, N.; del Fresno, C.; Fernandes, S.; Calvo, E.; Conde-Garrosa, R.; Kerr, W. G.; Sancho, D. J.Immunol 2015, In Press. 8. Brooks, R.; Fuhler, G. M.; Iyer, S.; Smith, M. J.; Park, M. Y.; Paraiso, K. H.; Engelman, R. W.; Kerr, W. G. SHIP1 Inhibition Increases Immunoregulatory Capacity and Triggers Apoptosis of Hematopoietic Cancer Cells. J. Immunol. 2010, 184, 3582-3589.

S40